Genome-Wide Identiﬁcation and Expression Patterns of the F-box Family in Poplar under Salt Stress

: The F-box family exists in a wide variety of plants and plays an extremely important role in plant growth, development and stress responses. However, systematic studies of F-box family have not been reported in populus trichocarpa . In the present study, 245 PtrFBX proteins in total were identiﬁed, and a phylogenetic tree was constructed on the basis of their C-terminal conserved domains, which was divided into 16 groups (A–P). F-box proteins were located in 19 chromosomes and six scaffolds, and segmental duplication was main force for the evolution of the F-box family in poplar. Collinearity analysis was conducted between poplar and other species including Arabidopsis thaliana , Glycine max , Anemone vitifolia Buch, Oryza sativa and Zea mays , which indicated that poplar has a relatively close relationship with G. max . The promoter regions of PtrFBX genes mainly contain two kinds of cis -elements, including hormone-responsive elements and stress-related elements. Transcriptome analysis indicated that there were 82 differentially expressed PtrFBX genes (DEGs), among which 64 DEGs were in the roots, 17 in the leaves and 26 in the stems. In addition, a co-expression network analysis of four representative PtrFBX genes indicated that their co-expression gene sets were mainly involved in abiotic stress responses and complex physiological processes. Using bioinformatic methods, we explored the structure, evolution and expression pattern of F-box genes in poplar, which provided clues to the molecular function of F-box family members and the screening of salt-tolerant PtrFBX genes.


Introduction
Plants are subjected to a variety of challenges, including drought, salt, high or low temperature, heavy metals and so on. In particular, salt is one of the biggest threats, leading to soil salinization of 19.5% of agricultural land every year, and it is expected that 30% of agricultural land will be salinized by 2028 [1] and more than 50% will be affected by salinization by 2050 [2]. So far, the genetic engineering technology is regarded as one of the most reliable and effective methods of developing salt-tolerant plants [3].
The ubiquitin/26S proteasome system (UPS) is an extremely important intracellular regulatory network to maintain the normal life activities of plants after receiving external stimulus signals. It achieves the purpose of pre-transcriptional regulation by selectively recognizing specific substrates and degrading the target proteins [4]. It involves three consecutive steps: (1) activating ubiquitin with the E1 enzyme, (2) transferring the activated ubiquitin to the E2 enzyme and (3) transporting ubiquitin to the E3 enzyme to degrade the target proteins. The SCF complexes are composed of SKP1, CUL1 and the F-box proteins that are main activators of E3 ligase [5]. As one component of the SCF complex, the F-box members are responsible for specific recognition and degradation of the target proteins, which may take part in different biological pathways [6].
F-box proteins with the representative HMM seeds from Pfam database were obtained from the Phytozome database. The conserved F-box domain was located at the N-terminal of the F-box proteins in poplar. All candidate proteins were screened by the SMART and Pfam databases for removing the proteins without an F-box domain and all redundant proteins. Finally, 245 members with the F-box domain were identified and used for our subsequent analysis (Table S1). According to their location on the poplar chromosomes, the 245 genes were numbered as PtrFBX1-PtrFBX245.
In order to understand the biological pathway involved in the F-box family in poplar, the functions of the 245 F-box proteins were predicted by EggNOG database. The results showed that most F-box genes played crucial roles in metabolic processes (46) and biological regulation (38) (Figure S1). Other F-box genes were involved in many essential biological processes such as the response to stimulus, various signaling pathways, growth and development, protein transport and protein modification. The Benjamin-Hochberg method was used to verify some important GO enrichment contents, including the cellular responses to hormone stimuli (GO:0032870), endogenous stimuli (GO:0071495) and auxin stimuli (GO:0071365); hormone-mediated signaling pathways (GO:0009755); responses to fungi (GO:0009620); pollen development (GO:0009555); multicellular organismal reproductive processes (GO:0048609); protein modification processes (GO:0036211) and the transferase complex (GO:1990234).

Phylogenetic Tree and Structure Analysis of the F-box Proteins in Poplar
In order to understand the evolutionary relationships of the F-box family in poplar, a phylogenetic tree with 245 full-length protein sequences was constructed by the neighbor-joining (NJ) method ( Figure 2). All proteins were divided into Groups A-P. Among these, Group O mainly contains the FBA and FBU subgroups, Group A is mainly composed of the FBU and FBD subgroups, Group L is mainly composed of the FBU subgroup, while Groups E, M and P are the smallest subgroups with only four members.

Phylogenetic Tree and Structure Analysis of the F-box Proteins in Poplar
In order to understand the evolutionary relationships of the F-box family in poplar, a phylogenetic tree with 245 full-length protein sequences was constructed by the neighborjoining (NJ) method ( Figure 2). All proteins were divided into Groups A-P. Among these, Group O mainly contains the FBA and FBU subgroups, Group A is mainly composed of the FBU and FBD subgroups, Group L is mainly composed of the FBU subgroup, while Groups E, M and P are the smallest subgroups with only four members. Furthermore, we analyzed the intron/exon structures and motifs of all proteins ( Figure 3). Each group shared similar structures and motifs. For example, 18 out of 28 members belonging to Group O have no intron and only one exon. Most members of Groups D and F have similar structures, that is, one exon and no intron. In addition, 20 conserved motifs in total were found in the PtrFBX proteins by MEME, and these motifs were further annotated by the SMART and Pfam databases (Table S2) Furthermore, we analyzed the intron/exon structures and motifs of all proteins ( Figure 3). Each group shared similar structures and motifs. For example, 18 out of 28 members belonging to Group O have no intron and only one exon. Most members of Groups D and F have similar structures, that is, one exon and no intron. In addition, 20 conserved motifs in total were found in the PtrFBX proteins by MEME, and these motifs were further annotated by the SMART and Pfam databases (

Chromosomal Location and Duplication Events of the F-box Family in Poplar
As shown in Figure 4a, all PtrFBX genes were distributed on all 19 chromosomes and six scaffolds. The largest number (32) of genes was on Chr1, followed by 20 genes on chromosome 6 and 8. There are 6, 8, 7 and 3 genes on Chr12, 15, 16 and 19, respectively, and there are 10 genes located on the scaffolds.  In addition, the collinearity of F-box genes between poplar and A. thaliana, G. max, A. vitifolia Buch, O. sativa and Z. mays was comparatively analyzed, and their collinearity maps were drawn by TBtools ( Figure 5). The results show that 56 PtrFBX genes in total are collinear with other species, and the collinear frequency is high on Chr2. Poplar has the most orthologous genes with G. max, as there are 53 PtrFBX genes collinear with 58 G. max genes; followed by A. vitifolia Buch, with 28 genes from A. vitifolia Buch being collinear with 31 PtrFBX genes. Poplar has fewer orthologous genes with A. thaliana, as only 10 PtrFBX genes are collinear with 10 A. thaliana genes (Table S3). It is noteworthy that there are four shared genes (PtrFBX40, PtrFBX41, PtrFBX48 and PtrFBX229) that are collinear with A. thaliana, G. max and A. vitifolia Buch. However, the collinearity relationship was Gene duplication events are essential for the evolution of family members [31]. Segmental duplication and tandem duplication are main driving forces of gene duplication. In order to understand the expansion of the F-box family in poplar, MCScanX was used to analyze their tandem duplication events [32]. In total, 27 tandem duplication genes were found, among which 23 genes were distributed on Chr1, 8, 10, 11, 15 and 17, and the others were located on the scaffolds. All tandem genes on Chr1, 8 and 17 all occurred in adjacent positions. As shown in Figure 4b, 38 genes had segmental duplication; these were distributed on 13 chromosomes (except Chr8, 10, 12, 15, 17 and 19), with six genes on Chr1 and Chr2. These results suggest that segmental duplication may play a critical role in the gene duplication events of the F-box family in poplar.
In addition, the collinearity of F-box genes between poplar and A. thaliana, G. max, A. vitifolia Buch, O. sativa and Z. mays was comparatively analyzed, and their collinearity maps were drawn by TBtools ( Figure 5). The results show that 56 PtrFBX genes in total are collinear with other species, and the collinear frequency is high on Chr2. Poplar has the most orthologous genes with G. max, as there are 53 PtrFBX genes collinear with 58 G. max genes; followed by A. vitifolia Buch, with 28 genes from A. vitifolia Buch being collinear with 31 PtrFBX genes. Poplar has fewer orthologous genes with A. thaliana, as only 10 PtrFBX genes are collinear with 10 A. thaliana genes (Table S3). It is noteworthy that there are four shared genes (PtrFBX40, PtrFBX41, PtrFBX48 and PtrFBX229) that are collinear with A. thaliana, G. max and A. vitifolia Buch. However, the collinearity relationship was not found between PtrFBX with O. sativa, and with Z. mays. In order to determine the selection pressure of the F-box family in poplar during evolution, the value of Ka/Ks was used to characterize the evolutionary ability of genes in genome-wide duplication events (Table S4). If we considering the case where there is no true value, there are 26 pairs of PtrFBX genes. Among these, the Ka/Ks scores of 25 PtrFBX genes are less than 1, ranging from 0.089402504 to 0.686712535. The results show that the In order to determine the selection pressure of the F-box family in poplar during evolution, the value of Ka/Ks was used to characterize the evolutionary ability of genes in genome-wide duplication events (Table S4). If we considering the case where there is no true value, there are 26 pairs of PtrFBX genes. Among these, the Ka/Ks scores of 25 PtrFBX genes are less than 1, ranging from 0.089402504 to 0.686712535. The results show that the majority of PtrFBX genes are limited in the evolutionary process and are mainly subject to strong purification selection, which is consistent with the report on F-box genes in soybean [9]. On the basis of the results of Ks, T = Ks/2r was used to estimate the approximate time of duplication events, which ranged from 52.78 to 124.61 Mya for segmental duplications and 31.72 to 462.33 Mya for tandem duplications.

Cis-Acting Elements Analysis of PtrFBX Promoter Sequences
Cis-acting elements are the key regions of intergenic regulation. Proteins can induce, repress and enhance gene transcription regulation in plants by binding to specific cis-acting elements [33]. To screen candidate genes that may be involved in the salt tolerance pathway, the upstream 2000 bp sequences of 245 PtrFBX genes were analyzed. Five hormonerelated and four stress-related components were found in the promoter regions of PtrFBX genes ( Figure S3). The hormone-related elements included P-box/TATC-box (response to gibberellin), TGA-element/AuxRR-core (response to auxin), ABRE (response to abscisic acid), the TCA element (response to salicylic acid) and the TGACG motif (response to jasmonic acid). Most PtrFBX promoters contained ABRE (172), which is an important regulatory element in response to ABA. Among 156 genes, there were 117 P-box and 39 TATC-box elements that respond to GA. There were two types of auxin-responsive elements, including 72 TGA elements and 23 AuxRR-core elements. As many as 104 genes also contained TCA elements and the TGACG motif. A few stress-related elements such as the WUN motif, the AT-rich element, MBS and LTR were also identified in the PtrFBX promoter regions.

Expression Pattern of PtrFBX Genes
After transcriptomic sequencing, 579.53 MB of raw data was obtained in total, and 531.7 MB of clean reads was obtained after filtration. The Q30 base percentage of each sample was above 98.36%, so it could be used for follow-up studies. Using RNA-Seq, we explored the expression patterns of PtrFBX genes in the roots, stems and leaves. In this study, 167 PtrFBX genes with FPKM ≥ 1 in any tissue were considered to be expressed ( Figure 6a). To explore expression patterns of F-box genes under salt stress in poplar, we profiled the mRNA abundance of DEGs across the leaves, stems and roots. As shown in Figure 6c-e, the results showed that 64 PtrFBX genes were DEGs in the roots, among which, 23 genes were significantly upregulated and 41 were downregulated. There were 26 DEGs in the stems (13 up-and 13 downregulated) and 17 in the leaves (11 up-and 6 downregulated). Among DEGs, there were eight shared genes between the leaves and roots, nine genes in the comparison of leaves and stems, and 12 shared between the roots and stems (Figure 6b). It should be noted that PtrFBX1, PtrFBX38, PtrFBX167 and PtrFBX168 were DEGs found across three tissues under salt stress, according to the RNA-Seq analysis (Table S5). In order to verify the accuracy of the RNA-Seq results, 23 DEGs were randomly selected for qRT-PCR (Table S6). The results showed that the expression trends of most DEGs were consistent between qRT-PCR and RNA-Seq (Figure 7), indicating the accuracy of RNA-Seq. In addition, a spatiotemporal expression analysis of four shared DEGs in the leaves and roots was performed ( Figure S4). The results showed that the expression trends of the four genes were similar and were induced, to some extent, by salt stress. In the leaves, the expression levels of the PtrFBX1, PtFBX168 and PtrFBX228 all reached their peaks at 12 h, and PtrFBX38 reached the highest level at 24 h, then their expression levels decreased. In the roots, the expression levels of PtrFBX38, PtrFBX168 and PtrFBX228 continued to increase. PtrFBX38 and PtrFBX168 reached the highest level at

Co-Expression Network Analysis
In a co-expression network, the genes in the same cluster may display similar functions in a specific pathway, and the unknown genes can be further identified by referring to similar genes in the same pathway. In the study, we selected three DEGs (PtrFBX1, PtrFBX38 and PtrFBX168) and one ABA response gene (PtrFBX228) to construct co-expression cluster networks by Spearman's method [34]. Among the four co-expression networks, the network centered on the PtrFBX228 gene was the largest (788 genes), while the one centered on PtrFBX1 was the smallest (272 genes) ( Figure S5).
In addition, the EggNOG database was used for gene enrichment analysis, and the top 30 GO terms with significant enrichment are displayed in Figure 8. The networks of the four genes are involved in diverse biological processes (Table S7)

Co-Expression Network Analysis
In a co-expression network, the genes in the same cluster may display similar functions in a specific pathway, and the unknown genes can be further identified by referring to similar genes in the same pathway. In the study, we selected three DEGs (PtrFBX1, PtrFBX38 and PtrFBX168) and one ABA response gene (PtrFBX228) to construct co-expression cluster networks by Spearman's method [34]. Among the four co-expression networks, the network centered on the PtrFBX228 gene was the largest (788 genes), while the one centered on PtrFBX1 was the smallest (272 genes) ( Figure S5).
In addition, the EggNOG database was used for gene enrichment analysis, and the top 30 GO terms with significant enrichment are displayed in Figure 8. The networks of the four genes are involved in diverse biological processes (Table S7)

Discussion
The F-box family is widely distributed in plants and acts as an important regulator in crucial cellular processes by selectively binding to specific substrates and then degrading the target genes within the ubiquitin/26S proteasome system [18]. F-box genes have been identified in many plant species such as Arabidopsis, rice, wheat, chickpea, etc. [12,34,35]. However, most of the F-box's function remain unknown in plants, especially in poplar. In this study, 245 F-box proteins were identified in poplar, with smaller parameters in the version of the poplar genome database Populus trichocarpa 3.0, which was inconsistent with former research [35]. According to the chromosomal distribution, gene structures, conserved motifs and characteristics of the phylogenetic tree of the F-box members, we divided the family into different subfamilies, analyzed the expression patterns of the F-box genes with and without salt stress in poplar using RNA-seq, and validated this by qRT-PCR. Representative genes were subjected to co-expression network analysis, and GO terms enrichment and comprehensive analyses were performed.
The release of the poplar genome makes it possible to conduct a comprehensive analysis of the evolution, structure and function of any family with complete reference sequences and annotations [29]. Many F-box family proteins actively participate in the protein degradation processes in specific pathways, mainly due to their diverse structural domains. Different domains will specifically interact with diverse substrates to perform different biological functions [12]. In this study, many conserved domains, such as LRR, Kelch, FBA, FBD, TUB, FBO (FIST, PAS-Kelch, Actin, etc.), and a combination of unknow

Discussion
The F-box family is widely distributed in plants and acts as an important regulator in crucial cellular processes by selectively binding to specific substrates and then degrading the target genes within the ubiquitin/26S proteasome system [18]. F-box genes have been identified in many plant species such as Arabidopsis, rice, wheat, chickpea, etc. [12,34,35]. However, most of the F-box's function remain unknown in plants, especially in poplar. In this study, 245 F-box proteins were identified in poplar, with smaller parameters in the version of the poplar genome database Populus trichocarpa 3.0, which was inconsistent with former research [35]. According to the chromosomal distribution, gene structures, conserved motifs and characteristics of the phylogenetic tree of the F-box members, we divided the family into different subfamilies, analyzed the expression patterns of the F-box genes with and without salt stress in poplar using RNA-seq, and validated this by qRT-PCR. Representative genes were subjected to co-expression network analysis, and GO terms enrichment and comprehensive analyses were performed.
The release of the poplar genome makes it possible to conduct a comprehensive analysis of the evolution, structure and function of any family with complete reference sequences and annotations [29]. Many F-box family proteins actively participate in the protein degradation processes in specific pathways, mainly due to their diverse structural domains. Different domains will specifically interact with diverse substrates to perform different biological functions [12]. In this study, many conserved domains, such as LRR, Kelch, FBA, FBD, TUB, FBO (FIST, PAS-Kelch, Actin, etc.), and a combination of unknow domains were found at the C-terminal of F-box proteins. LRR, Kelch, FBA, TUB and FBD are widely present in Arabidopsis, wheat and maize, whereas FBU (135 out 245) is considered to be the most abundant domain among different species [12,35]. The second most abundant domain varies greatly among different species. For example, FBD is the second most abundant in rice [12], while it is the FBA domain in poplar and wheat, which may participate in the degradation of target proteins [35].
The expansion of family members through large-scale duplication events is the basis for maintaining the stable existence of a large family in the process of evolution [31]. In order to better adapt to the changing environment, the protein families will expand through duplication events of the genome, such as bZIP [36], LEA [37] , XTH [38] etc.. As it is one of the largest protein families, duplication analysis of the F-box genes showed that 38 out of 245 genes are segmental duplication genes and 27 are tandem genes. The results suggest that segmental duplication contributed more to the expansion of the F-box family, which was also found in wheat [35]. More notably, the pattern of tandem duplication is biased among subgroups. Among the 27 tandem duplication genes, 18 genes belong to the FBU subgroup, which may reveal the direction of replication gene expansion. Ka/Ks values can be used to assess the evolutionary pressure on a gene family. There were 26 of 27 gene pairs with Ka/Ks < 1, indicating that most PtrFBX genes may have undergone strong purification selection during evolution. The differentiation of F-box duplication gene pairs can be estimated by the Ks, and the Ks values of segmental duplication and tandem duplication were about 52.78 to 124.61 Mya and 31.72 to 462.33 Mya, respectively. These details suggest that the differentiation of F-box family duplication was not synchronous. We further compared the collinearity relationship between poplar and five species, including three dicotyledons and two monocotyledons. Poplar has a better collinearity relationship with dicotyledons than with monocotyledons, among which PtrFBX genes have a best collinearity relationship with G. max.
Cis-acting elements function as important regulators in the transcription of neighboring genes. This study reported many cis-elements, such as ABRE, P-box, TATC-box, TGA-element, MBS, LRT, AT-rich, etc., that were found on the promoter regions of PtrFBX genes. These could be divided to two kinds, including hormone response elements and stress-related elements. Among these, ABRE is the most abundant hormone response element in F-box genes (70.2%). EDL3 has been reported to be ABA-induced and contributes to positive regulation of the root-to-flower transition in the ABA signaling pathway in Arabidopsis [39]. TC-repeat is the most abundant stress-related element, which is involved in immunity responses and plays an important role in the development of plant cells [40]. The WUN-motif functions in the process of wounding [41]. MBS is an important binding site of MYB transcription factors and regulates downstream genes through mutual binding under drought stress [42]. LRT is an important response element under low temperatures [43]. These details suggest that poplar's F-box genes may be involved in plant growth and abiotic stress responses by interacting with other genes.
As an important component of the SCF complex, the F-box family plays a vital role in plant growth, development, signal transduction, aging and senescence [44]. In Arabidopsis, F-box genes are vital for maintaining intracellular homeostasis through the auxin, JA and ABA pathways. The phylogenetic analysis of PtrFBX genes is helpful for screening similar functional F-box genes from other species. For example, the homologous gene of PtrFBX196 is TIR1, which encodes an auxin receptor and is involved in auxin signal transduction and transcription [45]. The homologous gene of PtrFBX61, AtCOI1, has been reported to participate in specific protein degradation and to combine with JAZ1 to form SCF [46]. The homologous gene of PtrFBX38 is MAX2, which is sensitive to ABA and can reduce stomatal closure, thus improving plant tolerance [20].
When plants are subjected to stress stimuli, they make a response through the corresponding life activities, such as signal pathway transduction and gene transcriptional regulation, so as to protect their normal intracellular activities. Transcriptome data provides a new method for exploring expression patterns of the relevant genes. The significant DEGs under the stress treatment may be involved in corresponding pathways. In this study, we explored the expression patterns of PtrFBX genes without and with salt stress. There were 82 DEGs in the three tissues in total, including 64 DEGs in the roots, 26 in the stems and 17 in the leaves. PtrFBX1, PtrFBX38 and PtrFBX168 were significantly upregulated in the three tissues. PtrFBX167 was only upregulated in the stems and was downregulated in the leaves and roots. To better understand the function of these DEGs, we identified their homologous genes in the TAIR database (https://www.arabidopsis.org/ (accessed on 10 August 2021)). For example, the homologous gene of PtrFBX12, PtrFBX52 and PtrFBX54 is AT1G273401, which can enhance tolerance to drought and salt in Arabidopsis [47]. At2G47900.1, which is homologous to PtrFBX228, can enhance ABA biosynthesis to promote bacterial susceptibility [48]. The homologous gene of PtrFBX143 is AT4G24210.1, which acts as a negative regulator and can antagonize the GA signal in the GA pathway [49]. Similarly, AT1G68050.1, the homolog of PtrFBX121, has been reported to negatively regulate DELLA proteins to promote flowering during long daylight cycles [50]. It has been reported that AT1G15670.1 is a negative regulator of cytokinin, and is the homologous gene of PtrFBX1 [51].
In the present study, we also conducted a co-expression network analysis of four interesting genes including three DEGs and one ABA-related response gene. These genes were annotated to be involved in abiotic stress responses and complex physiological processes. The gene networks of both PtrFBX168 and PtrFBX228 are significantly enriched in the responses to stimuli, biological regulation and responses to stress. The PtrFBX1 gene sets mainly focus on cell metabolic processes and responses to oxygen-containing compounds, while the PtrFBX38 gene sets cover the cell periphery and biological processes involved in interspecies interactions between organisms. The results indicate that different regulators operate complex protein regulatory networks.

Identification of F-box Proteins and GO Functional Annotation in Populus Trichocarpa
The ). The HMM Search program with a threshold of ≤1 × 10 −10 (https://www.ebi.ac.uk/Tools/hmmer/search/hmmsearch (accessed on 20 June 2021)) was applied to identify the candidate proteins. The SMART website (http://smart.embl.de/ (accessed on 22 June 2021)) and Pfam database were used to verify putative proteins. In addition, the EggNOG database (http://eggnog-mapper.embl.de/ (accessed on 25 June 2021)) was used for functional prediction of the PtrFBX genes and for gene ontology (GO) enrichment analysis. The GO enrichment results were displayed by TBtools [52].

Multiple Sequence Alignment and Phylogenetic Analysis
To explore the evolutionary relationships of the F-box family, 245 PtrFBX proteins with full-length sequences were obtained, and ClustalW software was used for multiple sequence alignment [53]. MEGA-X was used for construction of the phylogenetic tree by the neighbor-joining (NJ) method [54], and the parameters were set as follows: 1000 iterations of the bootstrap values; JTT (Jones-Taylor-Thornton) + G (gamma distributed) model; optional partial deletion of 95% of the threshold. Evolview online software (http://www. evolgenius.info/evolview/#/treeview (accessed on 5 July 2021)) was used for visualization.

Gene Sequence Analysis
SMART and the Pfam database were used to identify the conserved F-box domain located at the N-terminal of putative F-box proteins in poplar. According to multiple sequence alignment, the F-box domain logo was generated by Weblog (http://weblogo. berkeley.edu/logo.cgi (accessed on 5 July 2021)). On the basis of the highly conserved domains at the C-terminal, F-box members can be further divided into different subfamilies. Furthermore, MEME (http://meme-suite.org/tools/meme (accessed on 5 July 2021)) was used to predict the conserved motifs, and the following parameters were set: Classic mode; zero or one occurrence per sequence (zoops); number of motifs: 20; optimum motif width, ≥6 and ≤50.

Analysis of Gene Locations, Duplication Events and Ka/Ks
The Multiple Collinearity Scan toolkit was used for analyzing the duplication events of the F-box family in poplar [32]. According to the GFF3 file of P. trichocarpa 3.0 in the Phytozome database, we obtained the detailed chromosome locations of each PtrFBX gene. On the basis of the tandem duplication calculated by MCScan, we used TBtools to map the corresponding chromosomes of the PtrFBX genes. The Advanced Circos package was used to visualize the duplication relationships of PtrFBX gene pairs [52]. To further verify the collinearity of the F-box family among different species, the protein sequences and GFF3 annotation files of the F-box family of Arabidopsis thaliana, Glycine max, Anemone vitifolia Buch, Oryza sativa and Zea mays were also downloaded from the Phytozome database. The Dual Synteny Plotter package was used to calculate and visualize the collinear relationships of homologous proteins between P. trichocarpa and other species. The Ka/Ks calculator was used to verify the occurrence of duplication events between duplication gene pairs. The values of Ka (nonsynonymous substitution rate), Ks (synonymous substitution rate) and Ka/Ks were calculated, and Ks was used to calculate the approximate duplication time via the formula T = Ks/2r, and the value of r was 1.5 × 10 −8 substitutions per site per year for poplar [55].

Cis-Element Prediction of F-box Promoters in Poplar
The upstream 2000 bp promoter sequences of the PtrFBX genes were obtained from the Phytozome database. The cis-elements were predicted by the online tool PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 15 July 2021)) and visualized by TBtools software.

Plant Materials, Treatment and Transcriptome Analysis
The di-haploid Populus simonii × Populus nigra was used for research in the State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, China. The P. simonii × P. nigra seedlings were first cultured on half-strength MS culture medium for 20 days under greenhouse conditions, 25°C with a 16:8 h light-dark cycle. Healthy seedlings at a similar growth stage were transplanted to pots filled with soil: vermiculite: perlite = 3:2:1 for 1 month. The soil-cultured seedlings were then treated with a 150 mM NaCl solution for 12 h, 24 h and 36 h, with water as a control. Twelve samples for 0 h and 24 h, including the leaves, stems and roots, with two biological replicates, were paired-end sequenced on the Illumina HiSeq 2500 platform with a 10× sequencing depth. Trimmomatic v0.30 and FastQC v0.10.1 were used to sequence the raw data for quality filtering and analysis [56,57]. Hisat v0.1.6 software was used to map the resulting clean data to the reference genome of Populus trichocarpa [58]. The roots, stems and leaves of samples without the salt treatment were used as the control group, and the roots, stems and leaves of the 150 mM salt treatment were used as the experimental group. The mRNA abundance of all PtrFBX genes is presented as FPKM (fragments per kilobase million). The DESeq2 package in R was used for screening differentially expressed genes (DEGs) with two standards: false discovery rate (FDR) ≤0.05 and log2 fold change (FC) ≥1 [59]. The heatmaps of DEGs were generated with TBtools software. A Venn diagram of the DEGs was constructed by VEENY 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/index.html (accessed on 18 July 2021)).

Validation of DEGs by qRT-PCR
In order to verify the accuracy of the RNA-Seq, the relative expression levels of 23 genes were verified by qRT-PCR. The Populus Actin gene was used as an internal reference [60]. The 2 −∆∆Ct method was used to determine the relative expression levels of each gene, based on the expression level of each gene without the salt treatment. The sequences of the qRT-PCR primers are listed in Table S6.

Co-Expression Network and GO Annotation
To further explore genes that may respond to salt stress, we used 12 samples of three tissues (leaves, roots, stems), including 6 untreated and 6 salt-stressed samples, and conducted a network analysis of four interesting genes by Spearman's method [34]. Cytoscape was used to draw the co-expression networks of the four genes [61]. The genes with a p-value of ≤0.05 and a correlation coefficient of ≥0.8 were considered to be coexpressed genes. The EggNOG database was used for gene ontology annotation of the gene sets, TBtools was used to display the top 30 GO enrichment figures, and the correlation p-value of each annotation pathway was corrected by the Benjamin-Hochberg method [62].

Conclusions
In this study, 245 F-box family members in total were identified in poplar, which were distributed on 19 chromosomes and six scaffolds. On the basis of the C-terminal domains, the F-box family was divided into 16 groups in poplar. The phylogenetic analysis indicated that F-box proteins in the same group shared similar structures and motifs. Segmental duplication was main driving force for the expansion of F-box family members in poplar, and the F-box family has been under strong purification selection in the process of evolution. The promoter regions of PtrFBX genes mainly contain two kinds of cis-elements, which are related to hormone and stress responses. In addition, the expression patterns of F-box genes in the roots, stems and leaves of poplar were profiled by RNA-Seq under salt treatment. There were 82 DEGs in the three tissues in total, including 64 DEGs in the roots, 26 in the stems and 17 in the leaves. Co-expression network analysis of four representative PtrFBX genes showed that their co-expressed genes were involved in abiotic stress responses and complex physiological processes. The comprehensive analysis of F-box genes in poplar will provide new insights for understanding the evolution of the F-box family and their functions in response to abiotic stresses.

Institutional Review Board Statement:
The di-haploid P. simonii × P. nigra specimens were provided by the State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, China. The study complied with the relevant institutional, national and international guidelines and legislation.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated or analyzed during this study are included in this published article and information files. Informed consent was obtained from all subjects involved in the study. The raw sequencing data used during this study have been deposited in the NCBI's SRA with the accession number SRP267437 (https://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP267 437 (accessed on 17 July 2021)).